Histories of Dermatan Sulfate Epimerase and Dermatan 4-O-Sulfotransferase from Discovery of Their Enzymes and Genes to Musculocontractural Ehlers-Danlos Syndrome

Dermatan sulfate (DS) and its proteoglycans are essential for the assembly of the extracellular matrix and cell signaling. Various transporters and biosynthetic enzymes for nucleotide sugars, glycosyltransferases, epimerase, and sulfotransferases, are involved in the biosynthesis of DS. Among these enzymes, dermatan sulfate epimerase (DSE) and dermatan 4-O-sulfotranserase (D4ST) are rate-limiting factors of DS biosynthesis. Pathogenic variants in human genes encoding DSE and D4ST cause the musculocontractural type of Ehlers-Danlos syndrome, characterized by tissue fragility, joint hypermobility, and skin hyperextensibility. DS-deficient mice exhibit perinatal lethality, myopathy-related phenotypes, thoracic kyphosis, vascular abnormalities, and skin fragility. These findings indicate that DS is essential for tissue development as well as homeostasis. This review focuses on the histories of DSE as well as D4ST, and their knockout mice as well as human congenital disorders.


GAG-Protein Linker Region
Initial biosynthesis of DS side chains is evoked by the construction of a comm GAG-protein linker region tetrasaccharide, GlcUA-Gal-Gal-Xy-O-Ser ( Figure 2) [1,22-2 XYLT1 and XYLT2 encode β-xylosyltransferases (XylT), which transfer a Xyl moiety fro uridine diphosphate (UDP)-Xyl to specific serine residues in the core proteins of PGs b synthesized in the endoplasmic reticulum and/or cis-Golgi compartments [36-4 B4GALT7 encodes β1,4-galactosyltransferase-I (GalT-I), which transfers a β4Gal moie Figure 1. Common GAG-linker region and repeating disaccharide region of DS and CS with their potential sulfation sites. The DS backbone is composed of IdoUA and GalNAc, whereas CS is a stereoisomer of DS that includes GlcUA instead of IdoUA. These carbohydrate residues may be sulfated at various positions indicated by "SO 3 − ". It should be noted that sulfation modification of the hydroxy group occurs with various combinations but not all the hydroxy groups. 4-O-Sulfation on GalNAc residues in both DS and CS predominantly occurs in mammals. The linkage region tetrasaccharide, IdoUA-Gal-Gal-Xyl-, was isolated from DS-PGs of bovine aorta [25].
from UDP-Gal to Xyl-O-Ser in the core proteins of PGs in the Golgi apparatus [37,4 GalT-I interacts with XylT, which has been demonstrated through co-immunoprec tion and the course purification of GalT-I [50,51]. B3GALT6 encodes β1,3-galactosyl ferase-II (GalT-II), which transfers a β3Gal moiety from UDP-Gal to Galβ1-4Xylβ1in the core proteins of PGs [45,46,52]. B3GAT3 encodes β1,3-glucuronosyltransfe (GlcAT-I), which transfers a β3GlcUA moiety from UDP-GlcUA to Galβ1-3Galβ1-4X O-Ser in the core proteins of PGs ( Figure 2) [53][54][55][56][57]. Biosynthetic pathway of DS side chain of PGs by diverse glycosyltransferases, epime and sulfotransferases. Following the biosynthesis of core proteins (green), the common GAG tein linker region, GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-, is constructed by XylT, GalT-I, GalT-I GlcAT-I on the specific Ser residue(s) of core proteins. After the formation of the linker region, droitin synthases build up the chondroitin backbone. Subsequently, the epimerization of G moieties as well as sulfation of mainly GalNAc and a small proportion of IdoUA residues ar lyzed by DSE as well as D4ST and UST, respectively. Each enzyme and its coding gene are ind by blue and red characters, respectively.

DSE
DSE (also known as chondroitin-glucuronate C5-epimerase) converts GlcUA into IdoUA by epimerization of the C-5 carboxy group of GlcUA moieties in the repeating disaccharide region of the chondroitin precursor chain, [-4GlcUAβ1-3GalNAcβ1-] n , to construct the disaccharide region of dermatan, [-4IdoUAα1-3GalNAcβ1-] n ( Figure 2). In the 1970s, Malmström et al. demonstrated that the formation of IdoUA from the GlcUA residue occurred at the polymer level together with sulfation using fibroblast extract [80], which suggests the requirement of sulfation for the epimerization of GlcUA. Furthermore, Malmström revealed an important feature of the epimerization of GlcUA into IdoUA: the release of H from C-5 of the target GlcUA [81]. The incorporation of 3 H into the GlcUA residue of chondroitin from 3 H 2 O, or the liberated 3 H, which was released from chondroitin with 5-3 H-labeled GlcUA residues, can be quantified by liquid scintillation counting [82][83][84]. Dermatan is a better substrate than chondroitin for DSE, from human embryonic fibroblasts, but not CS or DS [83,86]. Tiedemann and Malmström et al. demonstrated high and low levels of chondroitin-glucuronate C5-epimerase activity in human lung fibroblasts and bovine chondrocyte cultures from nasal cartilage, respectively, whereas high 4-O-sulfotransferase activity was detected in both cells [87]. These findings suggest that 4-O-sulfation on GalNAc residues in both DS and CS may block DSE to fix the configuration of GlcUA and IdoUA.
In 2006, Maccarana et al. identified the gene SART2, encoding chondroitin-glucuronate C5-epimerase [88]. The enzyme was purified from the bovine spleen, in which GlcUA C5-epimerase activity is the highest among various tissues examined. Partially purified proteins were subjected to mass spectrometry analysis after treatment with trypsin. SART2, an unknown protein expressed in cancer cells and tissues [91], was identified from the 89-kDa band. There is no orthologous gene in the nematode (Caenorhabditis elegans) or fruit fly (Drosophila melanogaster). This is consistent with the finding that there is no DS in these organisms [99][100][101]. The recombinant SART2 protein exhibited strong GlcUA C5-epimerase activity. They proposed renaming SART2 as chondroitin-glucuronate C5-epimerase or DS-epimerase [88]. Currently, DSE is widely utilized as the name of the enzyme and gene. Research by Maccarana and Malmström involving the long-term study of DSE has greatly contributed to the elucidation of the functions of DSE as well as DS, and understanding of the pathogenic mechanisms of Ehlers-Danlos syndrome (EDS) with mutations in DSE, as described below.
In 2009, Maccarana et al. generated Dse-deficient mice [102], which exhibited reductions in DSE activity as well as DS, a smaller body size, a kinked tail at birth, and thicker collagen fibrils in the dermis and hypodermis [102]. Furthermore, defects in the fetal abdominal wall, exencephaly, spina bifida, and the impaired migration of aortic smooth muscle cells were also detected in Dse -/mice [103,104]. Dse-deficient mice also showed a reduction in the migration of skin-derived dendritic cells from the skin to draining lymph nodes that is a prerequisite for encounter with antigens [105]. Consequently, the initiation of the cellular and humoral immune response was diminished in Dse −/− mice.
In 2016, Gouignard et al. generated Xenopus laevis with knockdown of Dse and Dsel using morpholino antisense oligonucleotides [106]. Although the knockdown of Dse did not affect the formation of early neural crest progenitors, the epimerase activity and extent of neural crest cell migration were decreased in the morphants of Dse, which led to a decrease in neural crest-derived craniofacial skeleton, melanocytes, and dorsal fin structures. On the other hand, the knockdown of Dsel did not show clear phenotype except for a 12% decrease in DSE activity [106].
DS and/or DS-PGs are elevated in malignant cells and in the stroma [107], which is in agreement with an increase in DSE [108]. Thelin et al. demonstrated that a human esophageal squamous cell carcinoma treated with DSE-shRNA exhibited reduced migration and invasion abilities in vitro. Furthermore, these cells exhibited reduced cellular interaction with hepatocyte growth factor, inhibition of pERK1/2 signaling, and diminished focal adhesion formation and actin cytoskeleton dynamics [108]. These findings suggest that DS may contribute to the proliferation, invasion, and metastasis of esophageal squamous cell carcinoma via its binding with effector proteins such as hepatocyte growth factor and fibroblast growth factor at the cell surface and/or extracellular matrix.
In 2013, Müller et al. reported that EDS musculocontractural type 2 was caused by the homozygous mutation p.Ser268Leu in DSE (Tables 2 and 3) [109]. The clinical manifestations of their patient presented hypermobility of the finger, elbow, and knee joints, atrophic scars on skin, contracture of the thumbs and feet, and characteristic facial features [109]. DSE activities of recombinant p.Ser268Leu-DSE enzyme, as well as skin fibroblast cultures from the patient, were markedly weaker than in a respective control [109]. It should be noted that Mizumoto et al. developed a methodology to measure DSE activity without using a radioisotope such as 5-3 H-labeled GlcUA in chondroitin and 3 H 2 O. Namely, it utilized a CS-specific degrading enzyme, chondroitinase AC, fluorescent labelling, and an anion-exchange HPLC, based on the previous finding that dermatan is a better substrate than chondroitin for DSE [83], and that chondroitinase AC can act on a non-sulfated disaccharide unit, -GlcUA-GalNAc-, but that chondroitinase B, a DS-specific degrading enzyme, cannot act on a non-sulfated disaccharide unit, -IdoUA-GalNAc-. The reaction mixture containing the enzyme source, buffer, and desulfated DS (dermatan), was incubated at 37 • C. The chondroitin moiety of the reaction products with partial conversion from the IdoUA residue into the GlcUA residue can be digested with chondroitinase AC. The resultant di-or oligo-saccharides were labeled with a fluorophore at the reducing end, which was subjected to anion-exchange HPLC. When we analyzed the DSE activity and level of DS in the patient fibroblasts, significant decreases in the amount of DS disaccharide were detected in fibroblasts from the patient, accompanied by a decrease in the DSE activity, compared with those in fibroblasts from a healthy subject [109]. To date, nine cases have been reported as EDS musculocontractural type 2 caused by the mutation, p.Gly216Glufs*3, p.Arg267Gly, p.Ser268Leu, p.Tyr320*, p.Val333Cysfs*4, pPro384Trpfs*9, and pHis588Arg, in DSE [109][110][111][112][113].

DSEL/DSE2
In 2009, Pacheco et al. demonstrated that the second DSE, DSE2, encoded by DSEL, was identified as a homolog of DSE [90]. DSE2 consists of 1222 amino acids, which is larger than DSE (958 aa), has an epimerase domain,~700 amino acids, at the N-terminus, and a predicted O-sulfotransferase domain at the C-terminus [90]. DSE2/DSEL has epimerase activity, but no sulfotransferase activity toward desulfated DS, dermatan, and partially desulfated CS-A and CS-C [90]. The sulfotransferase domain in DSE2 has never been elucidated. It has been proposed as two epimerases, DS-epi1 and DS-epi2, encoded by DSE and DSEL genes, respectively [90]. To clarify the capacity of DSE and DSE2 in the formation of IdoUA in the DS chain, both genes were overexpressed in human embryonic kidney 293 cells. Overexpression of DSE led to the formation of IdoUA-block in both decorin and biglycan side chains, whereas overexpression of DSE2 resulted in the formation of IdoUA/GlcUA-hybrid structures, but not IdoUA-block [90]. Thus, tissues or cells such as the brain, in which DSEL is highly expressed more than DSE [114], may produce the CS/DS hybrid chain rather than a genuine DS chain. In fact, CS and DS chains in the mouse brain are predominantly present as hybrid structures containing both GlcUA and IdoUA residues in a single GAG chain [114][115][116].
In 2021, Bartolini et al. generated Dsel-deficient mice [117]. Although the Dsel -/mice showed reduced epimerase activity in the brain (89%), kidney (55%), spleen (44%), liver (38%), lung (34%), and skin (24% reduction) compared to that in wild-type tissues, Dsel -/mice exhibited no morphological, histological, or anatomical defects. The level of the IdoUA moiety in CS/DS from the newborn kidney and brain was reduced to 62 and 87% of wild-type mice, respectively, which may be consistent with the finding that DSEL was more predominantly expressed in the brain than DSE [91,92,117]. DSE may compensate for the loss of functions in DSEL.
In 2015, Stachtea et al. generated double knockout mice of Dse and Dsel [118]. They showed complete loss of DS, perinatal lethality, exencephaly, an umbilical hernia, and a kinked tail, suggesting that DS is essential for embryonic development.
In 2003, Goossens et al. reported that various homozygous or heterozygous mutations in DSEL, such as p.Val287Ile, p.Pro673Ser, p.Tyr730Cys, p.Pro942Ser, p.Ile1113Met, and a substitution of adenine to guanine in the 5 -non-coding region 546 bp upstream of the coding region, caused bipolar disorder [92]. In 2011, Shi et al. reported that a single nucleotide polymorphism, 75 kbp upstream of DSEL, caused a recurrent early-onset major depressive disorder [119]. Because DSEL, but not DSE, is predominantly expressed in the brain [91,92,117], the variants of DSEL may affect its neuronal functions.
In 2010, Zayed et al. reported that a congenital diaphragmatic hernia was caused by mutations in DSEL [120]. The probands showing diaphragmatic hernia have substitutions of amino acids: p.Met14Ile, p.Asn276Ser, p.Pro683Ser, p.Tyr740Cys, p.Thr842Ser, and p.Asp991Asn. Mouse Dsel was expressed in the murine diaphragm muscle on embryonic day (E) 13.5 by in situ hybridization using murine embryo sections [120]. Further analyses may be necessary to understand the pathogenic mechanisms leading to diaphragmatic hernia involving DS as well as DSEL.

CHST14/D4ST1
D4ST catalyzes the transfer of a sulfate group from an active sulfate, PAPS, to the C-4 hydroxy group of GalNAc residues in the repeating disaccharide region of dermatan, [IdoUA-GalNAc] n ( Figure 2). In 2001, Tiedemann et al. demonstrated that D4ST activity in human lung fibroblast and bovine articular chondrocyte cultures was higher than that in bovine nasal chondrocyte cultures [87]. Furthermore, Eklund et al. demonstrated that D4ST activity was detected in cell lysate from cultured human embryonic fibroblasts [93]. It has been demonstrated that even though the DSE reaction is freely reversible during the conversion of uronic acids on chondroitin as well as dermatan, neither CS nor DS is recognized by DSE [83]. They proposed that the formation of IdoUA during biosynthesis of DS might be enhanced by 4-O-sulfation of the GalNAc residue, thereby being locked in the configuration of IdoUA [87] (Figure 2), based on the protein sequence of purified C4ST from culture medium of rat chondrosarcoma after treatment with trypsin [121]. Subsequently, Hiraoka and Kang et al. identified CHST12 and CHST13, encoding C4ST-2 and C4ST-3, respectively [122,123]. In 2000 and 2001, Baenziger and colleagues identified two genes, CHST8 and CHST9, encoding GalNAc4ST-1 and GalNAc4ST-2, respectively, which transfer a sulfate group from PAPS to a terminal β1,4-linked GalNAc residue in GalNAcβ1-4GlcNAc-on N-linked oligosaccharides present on the glycoprotein hormones thyrotropin and lutropin [124,125].
In 2013, Akyüz et al. demonstrated that Chst14 -/mice showed a higher proliferation rate and longer cell processes of cultured Schwann cells from nerves and dorsal roots, compared with those of wild-type mice [131]. Although the Chst14 -/mice were fertile, one third of the knockout mice died between E16.5 and E18.5 and/or within a few days after birth [131]. The weights of the kidney, liver, heart, tibia, and whole body, but not brain, were reduced in the Chst14 -/mice compared with wild-type littermates. These findings suggest that D4ST-1 and/or DS may be essential for early tissue development.
In 2018, Yoshizawa et al. demonstrated a significantly decreased amount of DS, an abnormal structure of the basement membrane of capillaries, an alteration in the vascular structure with ischemic and/or necrotic-like change, and a reduced weight of the placenta in the placenta derived from Chst14 -/mice, compared with wild-types [132]. These findings indicate that D4ST-1 and DS may be indispensable for placental vascular development, and help to elucidate several cases of perinatal lethality caused by a mutation in CHST14 [133].
In 2021, Hirose et al. demonstrated that DS exhibited a round conformation with wrapped collagen fibrils in wild-type mice, whereas rod-shaped linear CS side chains of decorin observed at one end of collagen fibrils protruded outside the fibrils in Chst14 -/mice [134]. Furthermore, the skin tensile strength of Chst14 -/mice was lower than that of wild-type mice. These findings indicate that the DS side chain of decorin plays roles in the architecture of collagen fibrils and supporting skin strength.
In 2021, Nitahara-Kasahara et al. demonstrated that elephant teeth, thoracic kyphosis, increased skin fragility, and a kinked tail were observed in Chst14 -/mice [135,136]. Moreover, they also found a reduction in DS disaccharide in muscle and myopathy-related phenotypes including spread of the muscle interstitium and variation in fiber size, which may lead to decreased exercise capacity and lower grip strength in Chst14 -/mice. These findings suggest that D4ST-1 and/or DS is essential for the myogenesis of skeletal muscle.

Human Genetic Disorders Caused by Mutations in CHST14/D4ST1
In 2009, Dünder et al. reported that adducted thumb-clubfoot syndrome in 11 probands from four families exhibited the homozygous mutations p.Val49*, p.Arg213Pro, and p.Tyr293Cys, and the complex heterozygous mutation p.Arg135Gly/p.Leu137Gln in CHST14 [133]. Adducted thumb-clubfoot syndrome is an autosomal recessive disorder characterized by congenital defects in the heart, kidneys, or intestines, connective tissue fragility with aging, thin and translucent skin, coagulopathy, facial clefting, a typical facial appearance, joint instability, and contracture of the thumbs and feet [137]. Five out of the eleven probands with adducted thumb-clubfoot syndrome died in early infancy or childhood. Furthermore, their studies showed a reduced amount of DS and an increase in CS in fibroblasts from a patient with a mutation of p.Arg213Pro in CHST14 compared to control subjects [133].
In 2005, Kosho et al. reported two probands with generalized joint laxity, scoliosis, fragility, hyperextensibility, readily bruisable skin, atrophic scars, hypotonia, recurrent hematomas, pectus excavatum, and a mild delay of gross motor development [138]. In view of these features, they proposed probands with EDS kyphoscoliosis type VIB. In 2010, Kosho et al. reported six unrelated patients with EDS characterized by progressive multisystem fragility, spinal deformi-ties, recurrent dislocations, progressive skin and joint laxities, multiple congenital contracture of the joints, and characteristic craniofacial features [139]. Subsequently, Miyake et al. identified mutations in CHST14 encoding D4ST-1 in six probands [140]. The compound heterozygous mutations p.Lys69*/p.Pro281Leu, p.Pro281Leu/p.Cys289Ser, and p.Pro281Leu/p.Tyr293Cys or homozygous mutation p.Pro281Leu were identified in these probands. The recombinant CHST14/D4ST-1 mutants p.Pro281Leu, p.Cys289Ser, and p.Tyr293Cys, which were expressed by COS-7 cells, showed markedly lower D4ST activity than the recombinant wild-type [140]. Consistent with this finding, skin fibroblast cultures from patients with the mutation p.Pro281Leu or p.Pro281Leu/p.Tyr293Cys also showed considerably weaker D4ST activity than healthy subjects [140]. Thus, these substitutions of amino acids affected the enzymatic activity of D4ST-1. Instead of dermatan, a CS chain was produced on the decorin core protein of skin fibroblasts from patients [140]. Because 4-O-sulfation in DS inhibits the enzymatic reaction of the conversion of IdoUA into GlcUA by DSE, the lack of 4-O-sulfation in DS side chains in probands causes the reverse epimerization reaction, the conversion of IdoUA to GlcUA in dermatan by DSE, resulting in the re-formation of chondroitin, which is sulfated by C4ST to form CS.
In 2019, Hirose et al. demonstrated the skin pathology of probands with the musculocontractural type of EDS, with both transmission electron microscopy-based cupromeronic blue staining to detect GAG chains and immunostaining of decorin [141]. Collagen fibrils were tightly and regularly assembled in controls; in contrast, they were dispersed in the affected papillary to reticular dermis. Moreover, DS side chains of decorin-PG from control subjects were curved, maintaining close contact with attached collagen fibrils, whereas affected GAGs, CS side chains of decorin-PGs, were linear, stretching from the outer surface of collagen fibrils to adjacent fibrils. These findings suggest that structural alterations of GAG side chains on decorin-PG lead to spatial disorganization of collagen networks. In 2017, Mizumoto et al. demonstrated the absence of urinary DS in patients with pathogenic variants in CHST14/D4ST1 by an anion-exchange HPLC in conjugation with the DS-degrading enzyme chondroitinase B [142]. This method indicates the usefulness of urinary disaccharide compositional analysis of DS chains as non-invasive screening for this disorder.

Conclusions
Recent studies on in vivo functions of DSE, DSEL/DSE2, and CHST14/D4ST-1 using their knockout mice and human cells from patients have partially clarified the pathogenic mechanisms of the disorder as well as the biological significance of DS side chains of DS-PGs. These findings are due to marked contributions by a large number of basic studies since the 1970s by glycobiologists, biochemists, clinicians, and geneticists. Additionally, the knockout mice of these genes help further our understanding of the musculocontractural type of EDS. Advancements in our understanding of the molecular mechanisms involving DS may be necessary for the development of therapeutic approaches and agents.